Embodiments of the present application relate to systems and methods for detection of binding between biological and/or synthetic (organic or inorganic) molecules. In particular, the systems and methods provided herein enable measurement of the binding affinity between different molecules in both label-free and label-based modalities, as well as automated detection of gradients in the immobilized species.
The detection of binding between two different biological molecules has revolutionized multiple areas of healthcare and diagnostics. There currently are a multitude of schemes to detect the binding between two molecules, which can be subdivided as being label-free or label-based.
Label-based detection is widely used in protein microarrays due to the common availability of reagents and simple instrument requirements. However, these labeling strategies often alter both surface characteristics and natural activities of the query molecule. Such label-based techniques include chromogenic detection, fluorescent-based detection, radio-active labeling, and chemiluminescense. In all of these techniques, one of the molecule-types is immobilized on a surface (the “substrate molecule”). Subsequently, another molecule (labeled or unlabeled, the “target molecule”) is allowed to interact with the substrate. To determine if the pair of molecules bind, a secondary molecule that also binds the target molecule may be introduced, and such secondary molecule may be labeled with a motif, non-limiting examples of which include fluorescent tags or substrates on which enzymes can change colors. Thus, the motif provides a means to evaluate whether an interaction has occurred, for example, by optically measuring the signal coming from the sample. If the target molecule is independently labeled, however, the secondary molecule is unnecessary. Because each of these techniques typically requires multiple wash cycles, label-based techniques generally are time-consuming. A common label-based technique is an ELISA, which can also be done as a function of time to provided kinetic information of the binding reactions.
Label-free techniques, unlike label-based techniques, are capable of directly detecting if a target molecule is bound to the substrate molecule without the use of a secondary molecule with a motif. There are multiple label-free techniques, the most common of which are either optical (i.e., using surface plasmon resonances (SPR)) or mechanical (i.e., using a quartz microbalance (QM) to detect changes in the vibration resonant frequency).
SPR techniques measure the change in the thickness or refractive index of biomaterials at the interface between metal surfaces, usually a thin gold film (50-100 nm) coated on a glass slide in an ambient medium. The test proteins are immobilized on the gold film, an unlabeled query protein is added, and the change in the angle of reflection of light caused by binding of the query protein to the immobilized protein is measured to characterize biomolecular interactions in real-time. The angle at which the minimum intensity of the reflected light is obtained is directly related to the amount of biomolecules bound to the gold film.
Variations of this technique and alternative optical techniques also are known, but all rely on the change of the effective index of refraction of the substrate upon binding of the target molecules to the substrate. Acoustic biosensors also allow for the label-free detection of molecules and analysis of binding events. The acoustic biosensors generally are based on quartz crystal resonators, commonly found in electronic devices such as watches, computers and televisions. In such systems, there is a linear relationship between the mass adsorbed to the surface and the resonant frequency of the crystal in air, vacuum, or liquids.
Although existing label-based and label-free techniques provide methods for evaluating interactions between molecules, these techniques are usually time-consuming, complex, and lack the necessary sensitivity to detect both specific and unspecific-binding. Thus, there remains a need for simplified and versatile systems and methods for evaluating molecular binding.